1 In vitro protective effects of frequently used traditionally in cancer

2 prevention in Thai traditional medicine: an ethnopharmacological study

3 Natchagorn Lumlerdkija,b , Ranida Boonraka, Suksalin Booranasubkajorna, Pravit 4 Akarasereenonta,c, Michael Heinrichb*

5 a Center of Applied Thai Traditional Medicine, Faculty of Medicine Siriraj Hospital, Mahidol 6 University, 2 Wanglang Road, Bangkoknoi, Bangkok 10700,

7 b Research Group Pharmacognosy and Phytotherapy, UCL School of Pharmacy, 29-39 8 Brunswick Square, London WC1N 1AX, UK

9 c Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 10 Wanglang Road, Bangkoknoi, Bangkok 10700, Thailand

11 *Corresponding author.

12 E-mail address: [email protected] (N. Lumlerdkij), [email protected] (R. 13 Boonrak), [email protected] (S. Booranasubkajorn), [email protected] (P. 14 Akarasereenont), [email protected] (M. Heinrich)

15 Abstract

16 Ethnopharmacological relevance: Thai traditional medicine (TTM) has been used widely in 17 cancer management in Thailand. Although several Thai medicinal plants were screened for 18 pharmacological activities related to cancer treatment, such evidence still suffers from the lack 19 of linking with TTM knowledge.

20 Aim of the study: To document knowledge and species used in cancer prevention in TTM and 21 to preliminary investigate pharmacological activities related to the documented knowledge of 22 twenty-six herbal drugs used in cancer/mareng prevention.

23 Methods: Fieldwork gathering data on TTM concept and herbal medicines used in cancer 24 prevention was performed with TTM practitioners across Thailand. Later, water and ethanol 25 extracts from twenty-six herbal drugs mentioned as being used in cancer prevention were 26 screened for their protective effect against tert-butyl hydroperoxide-induced cell death in 27 HepG2 cells. Then active extracts were investigated for their effects on NQO1 activity, 28 glutathione level, and safety in normal rat hepatocytes.

29 Results: The fieldwork helped in the development of TTM cancer prevention strategy and 30 possible experimental models to test the pharmacological activities of selected medicinal 31 plants. Fifteen extracts showed significant protective effect by restoring the cell viability 32 to 40 - 59.3%, which were comparable or better than the positive control EGCG. Among them, 33 ethanol extracts from S.rugata and T.laurifolia showed the most promising chemopreventive 34 properties by significantly increased NQO1 activity, restored GSH level from oxidative 35 damage, as well as showed non-toxic effect in normal rat hepatocytes.

36 Conclusion: TTM knowledge in cancer prevention was documented and used in the planning 37 of pharmacological experiment to study herbal medicines, especially in cancer, inflammation, 38 and other chronic diseases. The proposed strategy should be applied to in vivo and clinical 39 studies in order to further confirm the validity of such a strategy. Other traditional medical 40 systems that use integrated approaches could also apply our strategy to develop evidence that 41 supports a more rational uses in traditional medicine.

42 Keywords: cancer prevention, traditional medicine, rugata, Thunbergia laurifolia

43

44 1. Introduction

45 Cancer patients worldwide have increased their interests in using complementary or alternative 46 medicines for cancer care. It was reported that 9 - 81% of cancer patients mentioned the uses 47 of at least one type of complementary or alternative therapy, especially herbal medicines, after 48 their cancer diagnosis (Damery et al., 2011). In Thailand, Thai traditional medicine (TTM) is 49 an essential form of integrative medicine used by cancer patients. Generally, it is considered to 50 be beneficial, especially in pain relief. However, the use of TTM in cancer patients lacks a 51 systematic development of an evidence-based approach (Poonthananiwatkul et al., 2015). 52 Although cytotoxicity of Thai medicinal plants were reported (Itharat et al., 2004; Lee and 53 Houghton, 2005; Mahavorasirikul et al., 2010; Saetung et al., 2005), other pharmacological 54 activities related to cancer treatment and prevention in TTM are still needed for developing a 55 more rational use.

56 TTM is considered a holistic medical system focusing on maintaining the balance of the body, 57 especially of the four fundamental elements (dhātu si) which are dhātu din (earth), dhātu nam 58 (water), dhātu lom (wind), and dhātu fai (fire). When a person loses this balance, he/she will 59 become ill (Chokevivat and Chuthaputti, 2005). Maintaining the balance of the elements is the 60 main strategy for preventing illnesses.

61 In modern Thai, mareng is commonly used to refer to cancer. In TTM scriptures, it is also used 62 to refer to other diseases, which mostly are severe skin conditions (Foundation for the 63 Promotion of Thai Traditional Medicine and Ayurved Thamrong School Center of Applied 64 Thai Traditional Medicine, 2007). Therefore, mareng is not equal to cancer. We previously 65 studied the Thai concept of mareng and proposed for the first time five characteristics of cancer 66 in TTM and compared them to Western medical concepts. In the same report, we also proposed 67 that a TTM condition called krasai could involve oxidative stress (Lumlerdkij et al., 2018). 68 Oxidative stress has an important role in carcinogenesis. Elevated reactive oxygen species 69 (ROS) levels can initiate DNA damage, help cancer cells to acquire proliferative signals and 70 resist apoptosis, and promote the invasion, metastasis and angiogenesis (Fiaschi and Chiarugi, 71 2012). Therefore, oxidative stress can be an important target in cancer prevention in both 72 biomedical and TTM senses.

73 Antioxidant systems are important in the prevention from toxic substances and carcinogens. 74 NAD(P)H:quinone oxidoreductase 1 (NQO1) is involved in the defence against toxicity and 75 carcinogenicity of quinones (Ross et al., 2000). Glutathione plays a crucial role in the 76 detoxification of toxic or carcinogenic reactive metabolites. It also protects against superoxide 77 and hydrogen peroxide formed during the metabolism (DeLeve and Kaplowitz, 1991; Melino 78 et al., 2011). Therefore, we proposed that NQO1 and glutathione were possible 79 pharmacological mechanisms to remove waste from the body in TTM sense (Lumlerdkij et al., 80 2018).

81 The objectives of this study are to document knowledge and species used in cancer prevention 82 in TTM and to assess pharmacological activities related to the documented knowledge; 83 including cytotoxicity, protective effect against oxidative stress, and effects on glutathione and 84 NQO1 enzyme activities of twenty-six species used in cancer/mareng prevention.

85 2. Materials and methods

86 2.1. Materials

87 AlamarBlue and PrestoBlue were bought from Abd Serotec. Primary rat hepatocytes from 88 Sprague-Dawley rats (RTCP10, Lot number RS874), Dulbecco's Modified Eagle Medium 89 (DMEM), Foetal Bovine Serum (FBS), PBS, Penicillin-Streptomycin (10,000 U/mL), 90 Williams E Medium, dexamethasone, human recombinant insulin, GlutaMAXTM, and HEPES 91 were purchased from Life technologies. HepG2 cells (ACC No 85011430, Lot 11C013), (-)- 92 Epigallocatechin gallate (EGCG) (E4268), paclitaxel (T1912), Albumin from bovine serum 93 (A2058), β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate 94 (NADPH) (N1630), L-Glutathione reduced (G4251), 5-5'-dithiobis (2-nitrobenzoic acid) 95 (DTNB) (D218200), thiazolyl blue tetrazolium bromide (MTT) (M5655), glutathione 96 reductase from baker's yeast (S. cerevisiae) (G3664), 5-Sulfosalicylic acid (SSA) (S2130), 97 DMSO, Complete Mini protease inhibitor cocktail (11836170001), and other reagents were 98 from Sigma Aldrich. NADP monosodium salt (sc-202724) and dicoumarol (sc-205647A) were 99 purchased from Santa Cruz Biotechnology. Glucose 6-phosphate disodium salt was from Bio 100 Basic Canada Inc. Yeast glucose 6-phosphate dehydrogenase (J61181) and flavin adenine 101 dinucleotide disodium salt (FAD) (A14495) were from Alfa Aesar. RIPA lysis buffer 10X was 102 from Merck Millipore. DC Protein Assay kit (500-0116) was from Bio-Rad Laboratories, Inc.

103 2.2. Ethnopharmacological field survey

104 Interviews with 33 TTM practitioners were carried out during December 2013 – April 2014 in 105 different regions of Thailand. The core questions used were ‘can mareng be prevented?’ or 106 ‘how can we prevent mareng?’ The project was approved by the UCL Research Ethics 107 Committee, Project ID: 5068/001, and Siriraj Institutional Review Board (Thailand), Protocol 108 number 779/2556(EC4). Information on the interviews and further details on data collection 109 are given in (Lumlerdkij et al., 2018). It followed the guidelines for such research (Heinrich et 110 al., 2018).

111 Identification of voucher specimens was done by comparison with books, monographs, or 112 authentic specimens from botanical gardens with helps from experienced TTM practitioners 113 from Center of Applied Thai Traditional Medicine, Faculty of Medicine Siriraj Hospital, 114 Mahidol University, Thailand (CATTM). Imported crude drugs were identified by comparing 115 macroscopic features with authentic materials obtained from Sun Ten Pharmaceutical Co., 116 Ltd.. Some common food plants, such as ginger, garlic, shallot, and mung bean were not 117 collected. Plant voucher specimens were deposited at Faculty of Pharmacy, Mahidol 118 University, Bangkok, Thailand. The taxonomic validation of the species is based on 119 http://mpns.kew.org/mpns-portal/ and www.theplantlist.org.

120 2.3. Species selection for pharmacological studies

121 The first step was to list all the species used in cancer/mareng prevention. Then the species 122 which were not endangered species, can be sustainably supplied, were mentioned by at least 123 two informants, and have not been studied extensively with regards to their chemopreventive 124 activities were selected for further bioactivity assessment. 125 2.4. Preparation of plant extracts

126 Plant materials were washed with deionized water, oven-dried between 40-60 oC, and ground. 127 Water extracts were prepared according to Thai traditional methods for decoctions. Briefly, 60 128 g of herbal powder was boiled with 600 ml of water until the volume reached about 200 ml. 129 The decoction was filtered through No.1 and No.4 filter paper (Whatman®) and then dried 130 using a freeze dryer. To prepare a 70% ethanolic extract, 60 g of herbal powder was added into 131 a glass bottle followed by 70% ethanol to cover the powder surface. The extraction was 132 performed for seven days with a 15-minutes shake every day. After 7 days, the extract was 133 filtered through No.1 and No.4 filter paper (Whatman®) and the solvent was then removed 134 using a rotary evaporator and a freeze dryer. The dry extracts were stored in a cool, dry place 135 and protected from light until use. Prior to cell-based assays, stock solutions of the extracts 136 were prepared using deionized water or DMSO. The water stock solutions were filtered through 137 0.22 μM syringe filter under a sterile condition. All stock solutions were kept at -20 oC until 138 use.

139 2.5. Cells

140 HepG2 cells were maintained in DMEM supplemented by 10% FBS and 1% 3 o 141 Penicillin/Streptomycin in 75 cm cell culture flasks at 37 C in 5% CO2/95% air. Fresh 142 complete medium was changed every three days. The cells were discarded after 15th 143 subculturing. Primary rat hepatocytes were thawed and maintained in collagen I-coated 96 well 144 plates. The thawing and plating medium was Williams E Medium supplemented with 5% FBS, 145 1 μM Dexamethasone, 1% Penicillin/Streptomycin, 4 μg/ml Human Recombinant Insulin, 2 146 mM GlutaMAXTM, and 15 mM HEPES, pH 7.4. The serum-free medium was refreshed every 147 24 hours to maintain the hepatocytes. The hepatocytes were discarded after five days.

148 2.6. Cytotoxicity of plant extracts in HepG2 cells

149 HepG2 cells (5,000 cells/well) were seeded into 96-well black plates and allowed to attach 150 overnight. The extracts (3.125 – 100 or 6.25 – 200 µg/ml), or paclitaxel (0.001 nM – 10 µM) 151 or EGCG (50 – 400 µM) as positive control, or fresh medium as control were then added. After 152 48 hours, 100 µl of diluted AlamarBlue solution (1:10 in complete medium) was replaced and 153 incubated for 2 hours at 37 oC. After that, the fluorescence intensity was measured at 560 nm 154 excitation and 590 nm emission using a microtiter plate reader (Infinite M200, Tecan). 155 Cytotoxicity was presented as % viability compared to the control. 156 2.7. Protective effect against oxidative stress-induced cell death

157 HepG2 cells (10,000 cells/well) were seeded into 96-well black plates. After 24 hours, the 158 medium was replaced with fresh complete medium as control, positive control (EGCG 50 µM), 159 and plant extracts at maximum non-toxic concentration (MNTC). After incubation with the 160 treatment for 24 hours, the medium was discarded and cell death was induced by addition of 161 200 µl of 0.5 mM t-BHP to each well. After 3 hours, AlamarBlue assay was performed to 162 determine the cell viability.

163 2.8. NQO1 activity assay

164 NQO1 activity was measured following (Fahey et al., 2004). Briefly, HepG2 cells (10,000 165 cells/well) were seeded into 96-well transparent plates and allowed to attach for one night. 166 Then the cells were incubated with extracts at MNTC or DMSO or menadione (positive 167 control) or dicoumarol (negative control). After the incubation, the cells were washed twice 168 with PBS. Then the cells were lysed with 30 µl of RIPA buffer supplemented with 1 mM PMSF 169 and shaken on a plate shaker for 20 minutes. Five µl of the cell lysate was transferred to a new 170 plate for quantification of total protein. Just before the addition, 1 ml of reaction mixture (500 171 μl of 0.5 M Tris-Cl, pH 7.4, 6.67 mg of Bovine serum albumin, 67 μl of 1.5% Tween-20, 6.7 172 μl of 7.5 mM FAD, 67 μl of 150 mM glucose 6-phosphate, 6 μl of 50 mM NADP, 20 units of 173 Yeast glucose 6-phosphate dehydrogenase, 3 mg of MTT, and fill deionized water to 10 ml) 174 was mixed with 1 μl of 50 mM menadione. Then 200 μl of the complete reaction mixture was 175 added to each well. The absorbance of the product was measured immediately at 610 nm and 176 every one minute up to five minutes with a microtiter plate reader (Infinite M200, Tecan). 177 NQO1 specific activity of treated cells were reported as percentage of the control (Prochaska, 178 1994).

179 2.9. Intracellular reduced glutathione (GSH) assay

180 Measurement of reduced form of glutathione (GSH) is based on the enzymatic recycling 181 method modified from (Allen et al., 2001). HepG2 cells (6 x 105 cells/well) were seeded into 182 6-well transparent plates and allowed to attach overnight. Then the cells were incubated with 183 extracts at MNTC or DMSO. After 24-hour incubation, the cells were treated with 0.7 mM t- 184 BHP for 4 hours. To prepare the cell lysate, the cells were washed twice with ice-cold PBS and 185 then lysed with 150 µl of ice-cold RIPA buffer containing cOmplete Mini tablets (150 µl of 186 7X cOmplete tablet stock solution was added to every 900 µl of RIPA buffer). Then the cells 187 were scraped off quickly and transferred to 1.5 ml reaction tubes and incubated in ice for 30 188 minutes. Then the tubes were ultrasonicated for 10 seconds and kept in ice for 10 seconds to 189 help lysing the cells completely. This step was repeated three times. The supernatant (cell 190 lysate) was transferred to new reaction tubes after centrifugation at 8000 xg for 10 minutes at 191 4 oC. The cell lysate was diluted with 5% SSA at 1:2 or 1:5 to precipitate proteins and to inhibit 192 γ-glutamyl transferase, which leads to the loss of GSH (Rahman et al., 2007). After 193 centrifugation at 10,000 xg for 10 minutes at 4 oC, 25 µl of supernatant was added to 96-well 194 plates (3 replicates/sample). Then 125 µl of ice-cold complete GSH reaction mixture (7.5 ml 195 of 143 mM Sodium Phosphate Buffer containing 6.3 mM EDTA, 1 ml of 2.39 mM NADPH 196 solution, 31.5 µl of glutathione reductase, and 500 μl of 0.01 M DTNB) was added to the 197 supernatant. The plates were then briefly shaken at 500 xg on a plate shaker. The absorbance 198 at 405 nm was immediately measured and every 30 seconds up to 5 minutes (11 cycles) by a 199 microtiter plate reader (Infinite M200, Tecan). A GSH standard curve (0.012 – 25 μM) was 200 performed together with each assay. Calculation of GSH levels in the samples were performed 201 according to (Allen et al., 2001).

202 2.10. Protein measurement

203 Protein measurement was performed using Bio-Rad DCTM Protein Assay kit. The absorbance 204 at 750 nm was measured with a microtiter plate reader (Infinite M200, Tecan). A BSA standard 205 curve was generated and used to quantify the amount of protein in cell lysate. The curve was 206 linear in the range of 0 – 1 mg/ml with R2 > 0.99.

207 2.11. Cytotoxicity of plant extracts in primary rat hepatocytes

208 The primary rat hepatocytes (20,000 cells/well) were seeded into collagen I-coated 96 well 209 plates and left for initial attachment for 6 hours. Then the hepatocytes were treated with CGe, 210 CHe, PS1e, TLe, or SR1e at various concentrations for 24 and 48 hours. Ethanol (0.0625 – 211 10% v/v) was used as a positive control and fresh serum-free medium served as control. After 212 the indicated incubation time, the medium was replaced by 10% PrestoBlue medium. The 213 fluorescent intensity was measured after 20 minutes at excitation wavelength of 535+9 nm and 214 emission wavelength of 590+20 nm. Cytotoxicity was presented as % viability compared to 215 the control.

216 2.12. Statistical analysis

217 Calculation of average, SD values, IC50 (the concentration of the extracts that inhibit the cell 218 viability for 50%), and maximum non-toxic concentration (MNTC) (the concentration of the 219 extracts that inhibit the cell viability for less than 20%), and one-way ANOVA analysis were 220 performed using GraphPad Prism 5.01 software (GraphPad Software, Inc.). All experiment 221 was performed at least N = 3. The level of significance was set at P < 0.05.

222 3. Results and discussion

223 3.1. Thai traditional medicine concept of cancer/mareng1 prevention

224 Twenty-nine informants suggested three methods for prevention of mareng which were taking 225 herbal medicines, eating proper food items (eg. local fresh vegetables), and life style 226 modification. Herbal medicines included multi-herbal preparations or single herbs. The uses of 227 herbal medicines were to remove waste from the body (detoxification), maintain the balance 228 of the four elements, nourish luead and namlueang, prevent krasai, and protect the liver (for 229 the details of luead, namlueang and krasai, see (Lumlerdkij et al., 2018)). In this study, we 230 focus on the detoxification and liver protection. The TTM practitioners suggested 231 detoxification for people with an increased risk, such as farmers and industrial workers who 232 continuously exposed to insecticides or lead or mercury. The TTM practitioners suggested 233 detoxification for people with an increased risk, such as farmers who used a large amount of 234 insecticides or industrial workers who were continuously exposed to lead or mercury. They 235 considered that it is important to protect the liver because the liver was among the most 236 important organs of the body. If the liver becomes abnormal, mareng could show up in many 237 organs, such as the liver itself, breast, uterus, ovary, or prostate gland. Figure 1 was developed 238 from the uses of preventive herbal medicines suggested by the informants. It shows our 239 representation of TTM cancer preventive strategy and related pharmacological assays (, i.e. an 240 etic interpretation).

241 The five characteristics of cancer and the uses of preventive herbal medicines suggested by the 242 informants assisted in developing the strategy for pharmacological assays of the herbal 243 medicines identified previously (Figure 1).

1 Since the cases mentioned by the informants had no medically confirmed diagnosis of cancer, the term ‘cancer/mareng’ is used throughout this report indicating that the data are based on reported uses. 244

245 Figure 1 Cancer prevention strategy from Thai traditional medicine and its possible pharmacological 246 models. Bioassay models for the four strategies; protection of liver, prevention from krasai, nourishment of 247 leaud & namlueang, and removal of waste, can be suggested. The balance of dhātu si is unclear in biomedical 248 sense. It is possible to involve with the cell homeostasis.

249

250 3.2. Selected plant samples for pharmacological assays

251 The informants mentioned 41 herbal remedies used in the prevention of cancer/mareng. A total 252 of 119 species belonging to 53 families were mentioned by TTM practitioners for their uses in 253 cancer/mareng prevention (Appendix). Five species could not be verified scientifically. 254 Species from the and Zingiberaceae were reported particularly frequently, with 11 % 255 and 7 % of total species, respectively. After the selection criteria were applied (Methods 2.3), 256 26 species were selected for further analyses. Table 1 shows frequency of citation (FC) of the 257 selected species reported to have preventive effects.

258 Table 1 Total samples in the pharmacological studies and their FC

No Scientific name Family Local name Part used Abbr. Voucher FC number 1 Allium ascalonicum L. Alliaceae Homdaeng young shoot AA - 2 (shallot) 2 Allium sativum L. Alliaceae Krathiam (garlic) young shoot AS - 3 No Scientific name Family Local name Part used Abbr. Voucher FC number 3 Aloe spp. Asphoderaceae Yadam processed resin AL NL-0028* 2 from leaf 4 Atractylodes lancea Asteraceae Kotkhamao dried rhizome AT NL-0029# 2 (Thunb.) DC. (atractylodes, Cang Zhu) 5 Capparis micracantha Capparidaceae Chingchi, root CM PBM05194 2 DC. Saemathalai 6 Citrus hystrix DC. Rutaceae Magrud (kaffir leaf CH - 2 lime) 7 Cladogynos orientalis Euphorbiaceae Chetphangkhi root CO PBM05196 3 Zipp. ex Span. 8 Coccinia grandis (L.) Cucurbitaceae Tamlueng (ivy whole plant CG PBM05195 2 Voigt gourd) 9 Derris scandens Fabaceae Thaowanpriang stem DS PBM05189 3 (Roxb.) Benth. (jewel vine) 10 Ferula assa-foetida L. Apiaceae Mahahing resin from root FA NL-0031* 3 (asafoetida) 11 Imperata cylindrical Poaceae Ya-kha (cogon root IC PBM05179 2 (L.) P.Beauv. grass)

12 Ligusticum striatum Apiaceae Kothuabua dried rhizome LS NL-0033# 2 DC. (L. Sinense Oliv. (Szechwan Cv. Chuanxiong) lovage, Chuan Xiong) 13 Peltophorum Fabaceae San-ngoen, insi twig PP PBM05180 2 pterocarpum (DC.) Backer ex K.Heyne 14 Piper ribesioides Wall. Piperaceae Sa-khan stem PA PBM05190 2

15 Piper retrofractum Piperaceae Dipli (long dried mature PR PBM05191 2 Vahl. pepper) unripe fruit 16 Piper sarmentosum Piperaceae Chaphlu whole plant PS1 PBM05181 Roxb. 3 17 Piper sarmentosum Piperaceae Chaphlu leaf PS2 PBM05181 Roxb. 18 Plumbaco indica L. Plumbaginaceae Chettamunploeng root PI PBM05192 3 daeng 19 Saussurea costus Asteraceae Kotkraduk dried root AU NL-0036# 2 (Falc.) Lipsch. (costus root, Mu Xiang)

Senegalia rugata § 20 (Lam.) Britton & Rose Leguminosae Sompoi (soap leaf SR1 NL-0037 pod) 2 Senegalia rugata § 21 (Lam.) Britton & Rose Leguminosae Sompoi (soap pod SR2 NL-0037 pod) 22 Smilax spp. Smilacaceae Khaoyennuea root SM NL-0038§ 2

23 Terminalia bellirica Combretaceae Samophiphek, fruit TB PBM05198 2 (Gaertn.) Roxb. Naeton (beleric myrobalan) 24 Thunbergia laurifolia Acanthaceae Rangchued (laurel leaf TL PBM05178 6 Lindl. clock vine, blue trumpet vine) 25 triandra Yanang stem TT PBM05193 3 (Colebr.) Diels 26 Tinospora crispa(L.) Menispermaceae Boraphet stem TC PBM05197 2 Hook. f. & Thomson (putarwali) 259 -, common species; #, the identification was performed by comparison of macroscopic features with authentic samples from Sun Ten and 260 Monographs of selected Thai Materia Medica Volume I; *, raw materials derived from plants; §; tentative identifications by comparison with 261 Monographs of selected Thai Materia Medica Volume I & II

262 3.3. Cytotoxicity of plant extracts in HepG2 cells

263 This assay was performed to determine MNTC of the plant extracts. Paclitaxel and EGCG had

264 IC50 values of 5.63 nM and 178.4 M, respectively. The MNTC value of EGCG was 119.3

265 M. Paclitaxel was used to validate the assay. EGCG, a well-known potential chemopreventive

266 agent (Landis-Piwowar and Iyer, 2014), was used as positive control. Table 2 shows IC50 and 267 MNTC values of all extracts. According to National Cancer Institute’s criteria, cytotoxicity of

268 plant extracts can be categorized into three groups; potent activity (log IC50 < 0), moderate

269 activity (0 < log IC50 < 1.10), and weak activity (1.10 < log IC50 < 1.5) (Fouche et al., 2008). 270 After cytotoxicity screening of 52 plant extracts, only SR2e exhibited moderate activity with

271 log IC50 = 0.85. A hit rate of 4.17% was obtained based on the number of species with moderate 272 activity expressed as a percentage of the 24 species tested. Interestingly, SR1e (ethanol extract 273 of SR leaves) did not show comparable effect with SR2e (ethanol extract of SR pods). Until 274 now, Senegalia rugata (syn.: Acacia concinna) has never been reported for its cytotoxicity in 275 HepG2 cells or anti-cancer activity before. Three extracts; SR2w, AUe, and ASe, showed weak

276 activity with log IC50 values of 1.16, 1.21, and 1.49, respectively. There was no report for 277 cytotoxicity in HepG2 for extract from young shoots of Allium ascalonicum (AS). Unlike SR 278 and AS, Saussurea costus (AU) was reported for anti-cancer activity of its isolated compounds; 279 alantolactone, isoalantolactone, and contunolide (Khan et al., 2013; Rasul et al., 2013a). 280 However, this study focused on chemopreventive properties rather than the ability to kill cancer 281 cells. Cytotoxicity assay was an important step in order to determine the MNTC as cell death 282 had to be avoided in other experiment.

283 Table 2 IC50 and MNTC values of plant extracts in HepG2 cells

No Extract IC50 (µg/ml) MNTC (µg/ml) codes Ethanol extract Water extract Ethanol extract Water extract (e) (w) (e) (w)

1 AA >200 >200 31.25 200

2 AS 31.04 >200 25.08 200

3 AL >200 >200 50 200

4 AT 51.57 >200 12.15 200

5 AU 16.19 >200 7.954 200 No Extract IC50 (µg/ml) MNTC (µg/ml) codes Ethanol extract Water extract Ethanol extract Water extract (e) (w) (e) (w)

6 CM >200 >200 200 200

7 CH 88.39 >200 50.17 200

8 CO 50.07 >200 24.71 200

9 CG >200 >200 100 100

10 DS 98.57 >200 65.84 150

11 FA 196.6 >200 12.5 200

12 IC 62.24 >200 33.12 200

13 LS 100.5 >200 36.02 200

14 PA >200 >200 44.44 200

15 PI 48.65 >200 37.06 89.81

16 PP 65.78 70.73 37.63 42.97

17 PR 154.3 >200 111.3 50

18 PS1 >200 >200 100 200

19 PS2 >200 >200 100 200

20 SR1 >200 >200 100 200

21 SR2 7.101 14.67 1.219 7.221

22 SM >200 >200 200 200

23 TB 115.0 198.5 10 23.85

24 TC >200 >200 150 200

25 TL >200 >200 50 200

26 TT 81.06 >200 53.47 18.04

284

285 3.4. Plant extracts with protective effect against oxidative stress-induced cell death

286 This assay was performed to screen for potential extracts with abilities to prevent oxidative 287 stress for further analysis. After a three hours-incubation with 0.5 mM t-BHP, the cell viability

288 was reduced to 31.47%. Pre-treatment with 50 M EGCG and 15 extracts significantly reduced

289 t-BHP-induced cell death (Figure 2). Water extract of TB at 30 g/ml (TBw30) showed the 290 most potent activity. It could restore the cell viability to 59.3%, which was higher than EGCG. 291 This might due to the antioxidant activity of the water extracts that showed comparable DPPH 292 radical scavenging activity to vitamin C (Chalise et al., 2010). Previous studies have reported 293 some activities which might contribute to the protective effect of these plant extracts. 294 Isoalantolactone isolated from AU activated Nrf2 (Rasul et al., 2013b). CH extracts exhibited 295 hydroxyl radicals scavenging activity and inhibited lipid peroxidation in HepG2 cells 296 (Laohavechvanich et al., 2010). Hydromethanolic extract of CG showed free radical 297 scavenging and antioxidant activities (Umamaheswari and Chatterjee, 2007). TL extracts 298 exhibited protective activity against ethanol-induced liver damage in rats and rat hepatocytes 299 (Pramyothin et al., 2005). Water extract of DS showed antioxidant effect (Laupattarakasem et 300 al., 2003). Ethanol extracts of the stem, leaf, and fruit and water extracts of the fruit and stem 301 of PS showed weak antioxidant activity in DPPH assay (Hussain et al., 2009). Therefore, its 302 protective effect might largely depend on other mechanisms. For AS, the protective effect 303 might due to the ability of organosulfur compounds and allyl derivatives in the induction of 304 GST, which is an important defensive enzyme (Bianchini, 2001). 305 Fifteen extracts that showed significant protective effect were then investigated in NQO1

306 activity assay. Commented [m1]: Will this figure be well visible in the MS?

307

308 Figure 2 Protective effects of plant extracts against tBHP-induced cell death. Cell viability of HepG2 cells 309 was measured by AlamarBlue assay after an induction of cell death by 0.5 mM t-BHP for three hours. One-way 310 ANOVA analysis showed that pre-treatment with 15 extracts for 24 hours significantly protected HepG2 cells 311 from t-BHP-induced cell death, *P < 0.05 (N >3). Where ‘w’ indicates water extract, ‘e’ indicates ethanol 312 extract, the number indicates the concentration of the extract tested.

313

314 3.5. Plant extracts induced NQO1 activity

315 We proposed that NQO1 was involved in the removal of waste from the body in TTM sense. 316 In this investigation, fifteen extracts that protected the cells from t-BHP; namely TBw, AUw, 317 ALw, CHe, AUe, CGe, TLe, TLw, TBe, DSw, SR1e, PIw, PS1e, ASw, and CMw, were tested 318 for the ability to induce NQO1 activity. After 72-hour incubation, five extracts; CHe, CGe, 319 TLe, SR1e, and PS1e, significantly increased NQO1 level in a dose-dependent manner (p < 320 0.05) (Figure 3). Dicoumarol (, an NQO1 inhibitor), reduced NQO1 activity by 50%. 321 Menadione, (an NQO1 inducer), enhanced NQO1 activity significantly in a dose-dependent 322 manner. The use of negative and positive controls showed that the assay was working properly. 323 0.1% and 0.2% DMSO, which were equal to the amount of DMSO in the extract treatment, did 324 not affect the enzyme activity. NQO1 activity induction might be one of the main protective 325 mechanisms of CHe, CGe, TLe, SR1e, and PS1e. Our result is in agreement with a previous 326 study which reported that T.laurifolia extracts induced NQO1 activity (Oonsivilai et al., 2007). 327 This is the first time that NQO1 induction activity of ethanol extracts from C.hystrix, C.grandis, 328 S.rugata, and P.sarmentosum was reported.

329 Even though potential cancer chemopreventive compounds must be proven to prevent tumour 330 induction in animal models or in clinical research, phase II enzyme assays in cell cultures have 331 been used for rapid screening ofwith such compounds. The induction of phase II enzymes, such 332 as GST and NQO1, is a major mechanism of a large number of anti-neoplastic and anti- 333 mutagenic agents (Prochaska, 1994). NQO1 is important for prevention from toxic quinones, 334 oxidative damage, and carcinogenesis (Nioi and Hayes, 2004). One of the protective actions of 335 NQO1 is scavenging of superoxide and superoxide-like radicals (Zhu et al., 2007). T-BHP 336 produces superoxide which results in cell damage (Slamenova et al., 2013). Therefore, 337 induction of NQO1 activity is relevant to the protective effect of the plant extracts against t- 338 BHP induced cell death, as well as helping to select potential candidates for the discovery of 339 chemopreventive agents. 340

341

342 Figure 3 NQO1 specific activity of HepG2 cells after 72 hour-treatment with indicated compounds (M)/ 343 extracts (g/ml). One-way ANOVA analysis showed the significant effect of tested substances, *P < 0.05 (N 344 >3). Where ‘e’ indicates ethanol extract and the number indicates the concentration of the extract tested.

345

346 3.6. Plant extracts restored glutathione level after t-BHP treatment

347 Similar to NQO1, we proposed that glutathione was also involved in the removal of waste from 348 the body. In this assay after the treatment with 0.7 mM t-BHP for 4 hours, GSH level of HepG2 349 cells dropped from 32.22 + 8.05 to 7.87 + 3.08 µM/ mg protein. Pre-treatment with SR1e and 350 TLe significantly restored GSH level in a dose-dependent manner (P < 0.05) (Figure 4). SR1e 351 50, 100 µg/ml, and TLe 50 µg/ml restored GSH level to 27.57 + 3.60, 30.85 + 4.88, and 22.72

352 + 4.89 µM/ mg protein, respectively, which were almost equal to the baseline (withoutno t- Formatted: Font: Italic 353 BHP). CHe also reversed the effect of t-BHP but not significantly. On the other hand, pre- 354 treatment with PS1e and high concentration of CGe reduced GSH level more than t-BHP 355 treatment alone, even though the effects were not significantly different. Prevention of the 356 depletion of GSH might be one of the main protective mechanisms of SR1e and TLe against 357 oxidative damage. T.laurifolia is well-known for its detoxifying properties and have shown 358 hepatoprotective activity in several rat models, as well as in cell cultures (Junsi and 359 Siripongvutikorn, 2016). However, this is the first time that ethanol extracts from S.rugata 360 (SR1e) and T.laurifolia (TLe) were reported for their ability to prevent GSH depletion by t- 361 BHP. 362 Glutathione has roles in the defence against oxidative stress, which is an important factor in 363 carcinogenesis. Many potential chemopreventive agents have been reported to induce GSH 364 level. For example, quercetin, a well-studied plant polyphenol found in onions, apples, berries, 365 tea, and red wine, showed the ability to increase GSH level, as well as to block the reduction 366 of GSH both in vivo and in vitro (Stagos et al., 2012). Therefore, GSH induction activity of the 367 ethanol extract from S.rugata (SR1e) and T.laurifolia (TLe) provide another evidence to 368 support their traditional uses in cancer prevention and their role as candidates for cancer 369 chemopreventive agent discovery.

370

371 Figure 4 GSH level after t-BHP treatment. One-way ANOVA analysis showed that pre-treatment with SR1e 372 and TLe for 24 hours significantly attenuated GSH depletion effect of t-BHP, *P < 0.05 compared to no pre- 373 treatment (N >3). Where ‘e’ indicates ethanol extract and the number indicates the concentration of the extract 374 tested. 375

376 3.7. The effects of plant extracts on cell viability in primary rat hepatocytes

377 The preliminary safety data of CHe, CGe, Ps1e, SR1e, and TLe (concentration 0 – 200 µg/ml) 378 were assessed by cytotoxicity assay in primary rat hepatocytes. While ethanol (0.06 – 10 % 379 V/V) reduced the cell viability to around 50%, the % viability of hepatocytes treated with the 380 extracts for 24 and 48 hours were between 77.8 – 104.52% and 73.1 – 102.38%, respectively.

381 The IC50 values of all extracts were more than 200 µg/ml. This shows that all five extracts were 382 not toxic to the hepatocytes (Figure 5). The cytotoxicity of these plant extracts in primary rat 383 hepatocytes has never been published before. In addition, Pramyothin et al. (2005) reported 384 that co-treatment of water extract from TL leaves at 2.5, 5.0 and 7.5 mg/ml with ethanol 385 significantly reduced the cell death of primary rat hepatocytes, compared to ethanol-treatment 386 alone (Pramyothin et al., 2005). This helps to confirm that TL extracts produced protective 387 effect rather than toxic effect in hepatocytes in in vitro.

388 389 Figure 5 Cytotoxicity of CGe, CHe, PS1e, SR1e, and TLe in primary rat hepatocytes. (A) after 24 hours- 390 incubation (B) after 48 hours-incubation (N > 3)

391

392 4. Conclusion

393 In this study, we successfully developed a strategy for the evaluation of pharmacological 394 activities based on TTM theory by using information from an ethnopharmacological fieldwork 395 (cf. Heinrich et al, 2019). In this study, we successfully developed a strategy going from 396 ethnopharmacological fieldwork to pharmacological experiments evaluating possible activities 397 based on TTM theory. This forms the foundation for a cancer prevention strategy based on 398 TTM concepts and its related pharmacological models as proposed here for the first time. We

399 found that S.rugata and T.laurifolia showed promising activities related to chemoprevention. Formatted: Font: (Default) Times New Roman, 12 pt 400 This could be additional information for using these herbs for preventive purposes. Our 401 findings are not only useful for TTM practitioners, but also serve as a scientific model to 402 investigate herbal medicine used in a cultural context for diseases, such as cancer, 403 inflammatory conditions, as well as many chronic diseases. In the future, chemical profiles of 404 the potential extracts should be studied in order to provide morea better understandings in the 405 biological effects. Furthermore, tthe strategy should be applied to in vivo and clinical studies 406 in order to further confirm the validity of such a strategy. . This is an approach which could Formatted: Font: 12 pt 407 serve as a model for developing an evidence based of oOther traditional medical systemsines Formatted: Font: 12 pt 408 that use holistic approach can also apply our strategy to by developinmg evidence that supports Formatted: Font: 12 pt 409 more rational uses inof specific traditional medicine.Other traditional medical systems that use Formatted: Font: 12 pt Formatted: Font: 12 pt Formatted: Font: 12 pt 410 holistic approach can also use our strategy as a model to develop traditional medicine with a 411 better evidence-base.

412 Acknowledgement

413 This study is supported by Faculty of Medicine Siriraj Hospital, Mahidol University, 414 Bangkok, Thailand (Grant number R015732024). The authors also thank Dr. Anthony Booker 415 for some authentic specimens.

416 Conflict of interest statement

417 The authors declare no conflict of interest.

418 Authors contributions

419 NL performed the fieldwork and all assays, analysed and interpreted the data, and wrote 420 the manuscript.

421 RB performed the fieldwork, prepared the voucher specimens and extracts.

422 SB collected the plants, prepared the voucher specimens and extracts.

423 PA participated in the manuscript preparation.

424 MH performed the fieldwork, analysed and interpreted the data, and wrote the 425 manuscript.

426 All authors read and approved the final manuscript.

427

428 References

429 Allen, S., Shea, J.M., Felmet, T., Gadra, J., Dehn, P.F., 2001. A kinetic microassay for 430 glutathione in cells plated on 96-well microtiter plates. Methods Cell Sci. 22, 305-312. 431 Bianchini, F., 2001. Allium vegetables and organosulfur compounds: do they help prevent 432 cancer? Environ. Health Perspect. 109, 893-902. 433 Chalise, J., Acharya, K., Gurung, N., Bhusal, R., Gurung, R., Skalko Basnet, N., Basnet, P., 434 2010. Antioxidant activity and polyphenol content in edible wild fruits from Nepal. Int. J. Food 435 Sci. Nutr. 61, 425-432. 436 Chokevivat, V., Chuthaputti, A., 2005. The role of Thai traditional medicine in health 437 promotion, The 6th Global Conference on Health Promotion, Bangkok, Thailand. 438 Damery, S., Gratus, C., Grieve, R., Warmington, S., Jones, J., Routledge, P., Greenfield, S., 439 Dowswell, G., Sherriff, J., Wilson, S., 2011. The use of herbal medicines by people with 440 cancer: a cross-sectional survey. Br. J. Cancer 104, 927. 441 DeLeve, L.D., Kaplowitz, N., 1991. Glutathione metabolism and its role in hepatotoxicity. 442 Pharmacol. Ther. 52, 287-305. 443 Fahey, J.W., Dinkova-Kostova, A.T., Stephenson, K.K., Talalay, P., 2004. The “Prochaska” 444 Microtiter Plate Bioassay for Inducers of NQO1, Methods Enzymol. Academic Press, pp. 243- 445 258. 446 Fiaschi, T., Chiarugi, P., 2012. Oxidative stress, tumor microenvironment, and metabolic 447 reprogramming: a diabolic liaison. Int. J. Cell Biol. 2012, 762825. 448 Fouche, G., Cragg, G.M., Pillay, P., Kolesnikova, N., Maharaj, V.J., Senabe, J., 2008. In vitro 449 anticancer screening of South African plants. J. Ethnopharmacol. 119, 455-461. 450 Foundation for the Promotion of Thai Traditional Medicine, Ayurved Thamrong School Center 451 of Applied Thai Traditional Medicine, 2007. Khamphi Takkasila, Tamra Kanphaet Thaidoem 452 (Phaetthayasat Songkhro Chabap Anurak) Volume 1, 2 ed. Supphawanit Kanphim, Bangkok.

453 Heinrich, M., A. Lardos; M. Leonti; C. Weckerle; M. Willcox with the ConSEFS advisory Formatted: Body Text 2, Indent: Left: 0 cm, First line: 0 454 group (Wendy Applequist, Ana Ladio, Chun Lin Long, Pulok Mukherjee, Gary Stafford) cm, Space Before: 6 pt 455 2018. Best Practice in Research: Consensus Statement on Ethnopharmacological Field 456 Studies – ConSEFS Journal of Ethnopharmacology 211 329-339. 457 http://dx.doi.org/10.1016/j.jep.2017.08.015

458 Heinrich, M.; Appendino, G.; Efferth, T., Fürst, R.; Izzo, A. A.; Kayser, O.; Pezzuto, J. M..; Formatted: Font: (Default) Times New Roman 459 Viljoen, A. 2019/2020 Best practice in research – overcoming common challenges in 460 phytopharmacological research. Journal of Ethnopharmacology 461 https://doi.org/10.1016/j.jep.2019.112230 462 Hussain, K., Ismail, Z., Sadikun, A., Ibrahim, P., 2009. Antioxidant, anti-TB activities, 463 phenolic and amide contents of standardised extracts of Piper sarmentosum Roxb. Nat Prod 464 Res 23, 238-249. 465 Itharat, A., Houghton, P.J., Eno-Amooquaye, E., Burke, P.J., Sampson, J.H., Raman, A., 2004. 466 In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer. J. 467 Ethnopharmacol. 90, 33-38. 468 Junsi, M., Siripongvutikorn, S., 2016. Thunbergia laurifolia, a traditional herbal tea of 469 Thailand: botanical, chemical composition, biological properties and processing influence. 470 International Food Research Journal 23, 923-927. 471 Khan, M., Li, T., Ahmad Khan, M., Rasul, A., Nawaz, F., Sun, M., Zheng, Y., Ma, T., 2013. 472 Alantolactone induces apoptosis in HepG2 cells through GSH depletion, inhibition of STAT3 473 activation, and mitochondrial dysfunction. BioMed Research International 2013, 719858. 474 Landis-Piwowar, K.R., Iyer, N.R., 2014. Cancer chemoprevention: current state of the art. 475 Cancer Growth Metastasis 7, 19-25. 476 Laohavechvanich, P., Muangnoi, C., Butryee, C., Kriengsinyos, W., 2010. Protective effect of 477 makrut lime leaf (Citrus hystrix) in HepG2 cells: implications for oxidative stress. Scienceasia 478 36, 112-117. 479 Laupattarakasem, P., Hoult, J.R.S., Itharat, A., Houghton, P.J., Hoult, J.R.S., 2003. An 480 evaluation of the activity related to inflammation of four plants used in Thailand to treat 481 arthritis. J. Ethnopharmacol. 85, 207-215. 482 Lee, C.C., Houghton, P., 2005. Cytotoxicity of plants from Malaysia and Thailand used 483 traditionally to treat cancer. J. Ethnopharmacol. 100, 237-243. 484 Lumlerdkij, N., Tantiwongse, J., Booranasubkajorn, S., Boonrak, R., Akarasereenont, P., 485 Laohapand, T., Heinrich, M., 2018. Understanding cancer and its treatment in Thai traditional 486 medicine: An ethnopharmacological-anthropological investigation. J. Ethnopharmacol. 216, 487 259-273. 488 Mahavorasirikul, W., Viyanant, V., Chaijaroenkul, W., Itharat, A., Na-Bangchang, K., 2010. 489 Cytotoxic activity of Thai medicinal plants against human cholangiocarcinoma, laryngeal and 490 hepatocarcinoma cells in vitro. BMC Complement. Altern. Med. 10, 55. 491 Melino, S., Sabelli, R., Paci, M., 2011. Allyl sulfur compounds and cellular detoxification 492 system: effects and perspectives in cancer therapy. Amino Acids 41, 103-112. 493 Nioi, P., Hayes, J.D., 2004. Contribution of NAD(P)H:quinone oxidoreductase 1 to protection 494 against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and 495 the arylhydrocarbon receptor basic helix-loop-helix transcription factors. Mutation 496 Research/Fundamental and Molecular Mechanisms of Mutagenesis 555, 149-171. 497 Oonsivilai, R., Cheng, C., Bomser, J., Ferruzzi, M.G., Ningsanond, S., 2007. Phytochemical 498 profiling and phase II enzyme-inducing properties of Thunbergia laurifolia Lindl. (RC) 499 extracts. J. Ethnopharmacol. 114, 300-306. 500 Poonthananiwatkul, B., Lim, R.H.M., Howard, R.L., Pibanpaknitee, P., Williamson, E.M., 501 2015. Traditional medicine use by cancer patients in Thailand. J. Ethnopharmacol. 168, 100- 502 107. 503 Pramyothin, P., Chirdchupunsare, H., Rungsipipat, A., Chaichantipyuth, C., 2005. 504 Hepatoprotective activity of Thunbergia laurifolia Linn extract in rats treated with ethanol: in 505 vitro and in vivo studies. J. Ethnopharmacol. 102, 408-411. 506 Prochaska, H.J., 1994. Screening strategies for the detection of anticarcinogenic enzyme 507 inducers. J. Nutr. Biochem. 5, 360-368. 508 Rahman, I., Kode, A., Biswas, S.K., 2007. Assay for quantitative determination of glutathione 509 and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1, 3159-3165. 510 Rasul, A., Bao, R., Malhi, M., Zhao, B., Tsuji, I., Li, J., Li, X., 2013a. Induction of apoptosis 511 by Costunolide in bladder cancer cells is mediated through ROS generation and mitochondrial 512 dysfunction. Molecules 18, 1418-1433. 513 Rasul, A., Khan, M., Ali, M., Li, J., Li, X., 2013b. Targeting apoptosis pathways in cancer with 514 alantolactone and isoalantolactone. The Scientific World Journal 2013, 248532. 515 Ross, D., Kepa, J., Winski, S., Beall, H., Anwar, A., Siegel, D., 2000. NAD(P)H:quinone 516 oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic 517 polymorphisms. Chem. Biol. Interact. 129, 77-97. 518 Saetung, A., Itharat, A., Dechsukum, C., Keawpradub, K., Wattanapiromsakul, C., 519 Ratanasuwan, P., 2005. Cytotoxic activity of Thai medicinal plants for cancer treatment. 520 Songklanakarin Journal of Science and Technology 7, 469-478. 521 Slamenova, D., Kozics, K., Hunakova, L., Melusova, M., Navarova, J., Horvathova, E., 2013. 522 Comparison of biological processes induced in HepG2 cells by tert-butyl hydroperoxide (t- 523 BHP) and hydroperoxide (H2O2): The influence of carvacrol. Mutation Research/Genetic 524 Toxicology and Environmental Mutagenesis 757, 15-22. 525 Stagos, D., Amoutzias, G.D., Matakos, A., Spyrou, A., Tsatsakis, A.M., Kouretas, D., 2012. 526 Chemoprevention of liver cancer by plant polyphenols. Food Chem. Toxicol. 50, 2155-2170. 527 Umamaheswari, M., Chatterjee, T.K., 2007. In vitro antioxidant activities of the fractions of 528 Coccinia grandis L. leaf extract. Afr J Tradit Complement Altern Med 5, 61-73. 529 Zhu, H., Jia, Z., Mahaney, J.E., Ross, D., Misra, H.P., Trush, M.A., Li, Y., 2007. The highly 530 expressed and inducible endogenous NAD(P)H:quinone oxidoreductase 1 in cardiovascular 531 cells acts as a potential superoxide Scavenger. Cardiovasc. Toxicol. 7, 202-211. 532

533

534

535

536

537

538

539

540

541

542

543

544 Appendix A. Medicinal plants mentioned for their uses in the prevention of cancer/mareng

No Local name (other name) Part used Tentative scientific name Family FC

1 Boraphet (putarwali) stem Tinospora crispa (L.) Hook. f. & Menispermaceae 2 Thomson 2 Buabok (Asiatic pennyworth, leaf Centella asiatica (L.) Urb. Apiaceae 1 gotu kola) 3 Buk (elephant yam) tuber Amorphophallus paeoniifolius Araceae 1 (Dennst.) Nicolson

4 Cha-em-thet (licorice) root Glycyrrhiza glabra L. Fabaceae 2

5 Chan (nutmeg ) flower, fruit Myristica fragrans Houtt. Myristicaceae 2

6 Cha-om (pennata wattle) root and twig (L.) Maslin Fabaceae 1

7 Chaphlu (wild betel) leave, whole Piper sarmentosum Roxb. Piperaceae 3 plant

8 Cheng-chu-chai (white aerial part Artemisia lactiflora Wall. ex DC. Asteraceae 1 mugwort Guizhou group)

9 Chetphangkhi root Cladogynos orientalis Zipp. ex Euphorbiaceae 3 Span. 10 Chettamunploengdaeng root Plumbaco indica L. Plumbaginaceae 3 (rose-coloured leadwort)

11 Chingchi, saemathalai root Capparis micracantha DC. Capparaceae 2 No Local name (other name) Part used Tentative scientific name Family FC

12 Dipli (long pepper) fruit Piper retrofractum Vahl Piperaceae 2

13 Dongdueng (climbing lily) root Gloriosa superba L. Colchicaceae 1

14 Fang (sappan tree) wood Caesalpinia sappan L. Fabaceae 1

15 Haewmu (nutgrass) rhizome Cyperus rotundus L. Cyperaceae 1

16 Hangnokyung daeng (red root Caesalpinia pulcherrima (L.) Sw. Fabaceae 1 flower) 17 Hangnokyung lueang (yellow root Caesalpinia pulcherrima (L.) Sw. Fabaceae 1 flower) 18 Hanumanprasankai leaf Schefflera leucantha R.Vig. Araliaceae 1

19 Homdaeng (shallot) young shoot Allium ascalonicum L. Amaryllidaceae 2

20 Huayang, thaowanyang stem Smilax ovalifolia Roxb. ex D.Don Smilacaceae 1 (kumarika) 21 Kamphaengchetchan wood Salacia chinensis L. Celastraceae 1

22 Kanphlu (clove) flower Syzygium aromaticum (L.) Merr. & Myrtaceae 1 L.M.Perry 23 Kaprao (holy basil) leaf Ocimum tenuiflorum L. Lamiaceae 1

24 Kasalong (Indian cork tree) stem bark Millingtonia hortensis L.f. Bignoniaceae 2

25 Katangbai (bandicoot berry) leaf Leea indica (Burm. f.) Merr. Vitaceae 1

26 Kha (galangal, Thai ginger) rhizome Alpinia galanga (L.) Willd. Zingiberaceae 1

27 Khamfoi (safflower) flower Carthamus tinctorius L. Asteraceae 1

28 Khaminkhruea, nae khruea root Arcangelisia flava (L.) Merr. Menispermaceae 1 (yellow-fruit moonseed)

29 Khanghuamu N/A N/A N/A 1

30 Khaotong, Phlukhao whole plant Houttuynia cordata Thunb. Saururaceae 1 (fishwort) 31 Khaoyennuea rhizome Smilax spp. Smilacaceae 2

32 Khaoyentai rhizome Smilax spp. Smilacaceae 2

33 Khaton root, wood Cinnamomum ilicioides A.Chev. Lauraceae 1

34 Khing (ginger) rhizome Zingiber officinale Roscoe Zingiberaceae 5

35 Khoklan wood Mallotus repandus (Rottler) Müll. Euphorbiaceae 1 Arg. 36 Khontha root Harrisonia perforata (Blanco) Rutaceae 1 Merr. 37 Kloi tuber Dioscorea hispida Dennst. Dioscoreaceae 2

38 Kothuabua, (Szechwan rhizome Ligusticum striatum DC. Apiaceae 2 lovage rhizome, Chuan Xiong) 39 Kotkamao (atractylodes, dried rhizome Atractylodes lancea (Thunb.) DC. Asteraceae 2 Cang Zhu) No Local name (other name) Part used Tentative scientific name Family FC

40 Kotkraduk (costus root, Mu dried root Saussurea costus (Falc.) Lipsch. Asteraceae 2 Xiang) 41 Kotphungpla, Samothai fruit Terminalia chebula Retz. Combretaceae 4 (terminalia gall, myrobalan gall) 42 Krachai (fingerroot) rhizome Boesenbergia rotunda (L.) Mansf. Zingiberaceae 1

43 Kradaddaeng (red giant taro) rhizome Alocasia macrorrhizos (L.) G.Don Araceae 1

44 Kradadkhao (white giant rhizome Alocasia macrorrhizos (L.) G.Don Araceae 1 taro) 45 Kradon (slow match tree) young leaf Careya arborea Roxb. Lecythidaceae 1

46 Kradukkaidam leaf Justicia fragilis Wall. Acanthacaea 1

47 Krathiam (garlic) young shoot Allium sativum Linn. Amaryllidaceae 3

48 Krawan (Siam cardamom) fruit Amomum compactum Sol. ex Zingiberaceae 1 Maton 49 Lamchiak, toei-ta-le root Pandanus odorifer (Forssk.) Pandanaceae 1 (umbrella tree) Kuntze 50 Maduea chumpon (cluster root Ficus racemosa L. Moraceae 1 fig) 51 Maduk root Siphonodon celastrineus Griff. Celastraceae 1

52 Mafai (Burmese grape) heartwood, Baccaurea ramiflora Lour. Phyllanthaceae 1 root, bark 53 Mafueang (carambola, heartwood, Averrhoa carambola L. Oxalidaceae 1 starfruit) root, bark 54 Magrud (kaffir lime) leaf Citrus hystrix DC. Rutaceae 2

55 Mahahing (asafoetida, oleo-gum- Ferula assa-foetida L. Apiaceae 3 stinking gum, devil’s dung) resin

56 Mahuad stem bark Lepisanthes rubiginosa (Roxb.) Sapindaceae 1 Leenh. 57 Makham (tamarind) leaf Tamarindus indica L. Fabaceae 1

58 Makhamkai leaf Putranjiva roxburghii Wall. Putranjivaceae 1

59 Makhampom (emblic fruit Phyllanthus emblica L. Phyllanthaceae 3 myrobalan) 60 Maklamtanu (crab's eye vine, sap wood Abrus precatorius L. Fabaceae 1 American pea) 61 Maliwanpa root N/A N/A 1

62 Manao (lime) leaf, juice Citrus × aurantiifolia (Christm.) Rutaceae 2 from fruits Swingle 63 Maprang (marian plum, heartwood, Bouea macrophylla Griff. Anacardiaceae 1 gandaria, plum ) root, bark 64 Mapring (Burmese plum, heartwood, Bouea oppositifolia (Roxb.) Adelb. Anacardiaceae 1 plum-mango) root, bark 65 Marum (horseradish tree, leaf Moringa oleifera Lam. Moringaceae 2 drumstick tree) 66 Matum (bael, golden apple) fruit Aegle marmelos (L.) Corrêa Rutaceae 2

67 Muakdaeng stem Wrightia coccinea (Roxb. ex Apocynaceae 1 Hornem.) Sims No Local name (other name) Part used Tentative scientific name Family FC

68 Muakkhao stem Wrightia pubescens subsp. Apocynaceae 1 pubescens 69 Ngueakplamo dokmuang leaf Acanthus ilicifolius L. Acanthaceae 1

70 Nontaiyak root tuber Stemona tuberosa Lour. Stemonaceae 1

71 Oi-dam (sugar cane) stem Saccharum officinarum L. Poaceae 1

72 Phakbungdaeng (morning root Ipomoea aquatica Forssk. Convulvulaceae 1 glory) 73 Phakchiangda (Gymnema young Gymnema inodorum (Lour.) Decne. Apocynaceae 1 sylvestre) flower, young leaf

74 Phakkradhuawaen (para young leaf Acmella caulirhiza Delile Asteraceae 1 cress) 75 Phakpaewdaeng whole plant Iresine diffusa f. herbstii (Hook.) Amaranthaceae 1 Pedersen 76 Phaktaew leaf Cratoxylum formosum (Jack) Hypericaceae 1 Benth. & Hook.f. ex Dyer 77 Phakwanban leaf Sauropus androgynus (L.) Mer. Phyllanthaceae 1

78 Phak-wan-pa leaf Melientha suavis Pierre Opiliaceae 1

79 Phitsanad root Sophora exigua Craib Fabaceae 1

80 Phrikpa, Phriknaiphran N/A N/A N/A 1

81 Phrikthai, black pepper fruit Piper nigrum L. Piperaceae 2

82 Pua-ki-nai (Huang Qin, root Scutellaria baicalensis Georgi Lamiaceae 1 Baikal skullcap) 83 Rangchued (laurel clock stem, root Thunbergia laurifolia Lindl. Acanthaceae 6 vine, blue trumpet vine) 84 Reo-noi fruit Amomum villosum Lour. Zingiberaceae 1

85 Kotnamtao (Rhubarb, Da rhizome Rheum Palmatum L., Polygonaceae 1 Huang) R. officinale Bail., R. tanguticum (Maxim. Ex Regel) Maxim. Ex Balf. 86 Rong, rongthong (gamboge) resin Garcinia hanburyi Hook.f. Clusiaceae 1

87 Sakhan stem Piper aff. pendulispicum C.DC. Piperaceae 2

88 Samodingu fruit Terminalia citrina (Gaertn.) Roxb. Combretaceae 1 ex Flem 89 Samophiphek, naeton fruit Terminalia bellirica (Gaertn.) Combretaceae 3 (beleric myrobalan) Roxb. 90 Samothet fruit Terminalia spp. Combretaceae 1

91 Sankham (Chinese albizia, twig Albizia chinensis (Osbeck) Merr. Fabaceae 2 silk tree) 92 San-ngoen, insi (copperpod, twig Peltophorum pterocarpum (DC.) Fabaceae 2 golden flamboyant) Backer ex K.Heyne

93 Somchin (mandarin orange) root Citrus × aurantium L. Rutaceae 1

94 Sompoi (soap pod) leaf, pod Senegalia rugata (Lam.) Britton & Fabaceae 2 Rose No Local name (other name) Part used Tentative scientific name Family FC

95 Ta-khlai (lemongrass) rhizome Cymbopogon citratus (DC.) Stapf Poaceae 1

96 Tamlueng (ivy gourd) whole plant Coccinia grandis (L.) Voigt Cucurbitaceae 2

97 Thao-en-on stem Cryptolepis dubia (Burm.f.) Asclepiadaceae 1 M.R.Almeida 98 Thaowandaeng stem Ventilago denticulata Willd. Rhamnaceae 1

99 Thaowanpriang (jewel vine) stem Derris scandens (Roxb.) Benth. Fabaceae 3

100 Thaoyaimom root Clerodendrum indicum (L.) Kuntze Lamiaceae 1

101 Thiandam (fennel flower, seed Nigella sativa L. Ranunculaceae 1 black caraway) 102 Thiankao (cumin) fruit Cuminum cyminum L. Apiaceae 1

103 Thua-phu (winged bean) root Psophocarpus tetragonolobus (L.) Fabaceae 1 DC. 104 Wan hokmokkhasak root N/A N/A 1

105 Wan khothongkae rhizome Curcuma sp. Zingiberaceae 2

106 Wan nakkharat, Wan rhizome Sanseviera zeylanica (L.) Willd. Asparagaceae 1 hangnak (Ceylon bowstring hemp, devil's tongue)

107 Wan thonmokkhasak rhizome Kaempferia sp. Zingiberaceae 1

108 Wanmahakan root Gynura hispida Thwaites Asteraceae 1

109 Wanphetchahueng (giant root Grammatophyllum speciosum Orchidaceae 1 orchid, tiger orchid) Blume

110 Wanphetchaklab rhizome Boesenbergia thorelii (Gagnep.) Zingiberaceae 1 Loes 111 Ya khaosan, Sanrangdid whole plant N/A N/A 1

112 Ya nuadmaew (cat's whisker) whole plant Orthosiphon aristatus (Blume) Lamiaceae 1 Miq. 113 Ya nuadruesi (black whole plant Heteropogon contortus (L.) Poaceae 1 speargrass, tanglehead) P.Beauv. ex Roem. & Schult.

114 Ya tudma whole plant Paederia pilifera Hook. f. Rubiaceae 1

115 Yadam dried latex Aloe spp. Asphodelaceae 2

116 Yakha (blady grass, cogon root Imperata cylindrica (L.) P.Beauv. Poaceae 2 grass) 117 Yanang root, stem (Colebr.) Diels Menispermaceae 3

118 Yapakkhwai (Egyptian whole plant Dactyloctenium aegyptium (L.) Poaceae 1 crowfoot grass) Willd. 119 Yo (Indian mulberry) fruit Morinda citrifolia L. Rubiaceae 2

545

546